Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10.

Antiviral signaling, immune response and cell metabolism are dysregulated by SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical roles in mitochondrial function and morphology. On the other hand, all four ORFs altered mitochondrial dynamics and function, but only ORF3a and ORF9c induced a marked alteration in mitochondrial cristae structure. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features both shared across all accessory proteins and specific for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response.

Infection with Salmonella Typhimurium (S. Typhimurium) is a common cause of food-borne zoonosis leading to acute gastroenteritis in humans and pigs, causing economic losses to producers and farmers, and generating a food security risk. In a previous study, we demonstrated that S. Typhimurium infection produces a severe transcriptional activation of inflammatory processes in ileum. However, little is known regarding how microRNAs regulate this response during infection. Here, small RNA sequencing was used to identify 28 miRNAs differentially expressed (DE) in ileum of S. Typhimurium-infected pigs, which potentially regulate 14 target genes involved in immune system processes such as regulation of cytokine production, monocyte chemotaxis, or cellular response to interferon gamma. Using in vitro functional and gain/loss of function (mimics/CRISPR-Cas system) approaches, we show that porcine miR-194a-5p (homologous to human miR-194-5p) regulates TLR4 gene expression, an important molecule involved in pathogen virulence, recognition and activation of innate immunity in Salmonella infection.

Salmonella Typhimurium Infection Along the Porcine Gastrointestinal Tract and Associated Lymphoid Tissues.

Salmonella is a major foodborne pathogen and pork is one of the main sources of human salmonellosis. Understanding the pathogenesis and progression of the infection within the host is of interest to establish potential approaches to control the disease in pigs. The present study evaluates factors such as intestinal colonization, fecal shedding, and pathogen persistence by 2 studies using experimental challenge with Salmonella Typhimurium in weaned pigs and euthanasia at different time points (1, 2, and 6 and 2, 14, and 30 days postinfection [dpi], respectively). Histopathology of intestine at early time points (1 dpi and 2 dpi) showed severe damage to the epithelium together with an increase in polymorphonuclear cells and macrophages (P < .001), particularly in jejunum and ileum. Large quantities of Salmonella were detected within the contents of the ileum, cecum, and colon in early infection. Salmonella could also be observed in the medulla of tonsils and mesenteric lymph nodes. From 6 dpi onward, signs of recovery were observed, with progressive restoration of the epithelium, reduction of the inflammatory infiltrate, and elimination of Salmonella from the mucosa. Concentration of Salmonella in feces and ileum content decreased, but shedding did not cease even at 4 weeks after infection. Persistence of the bacteria in mesenteric lymph nodes was identified within the connective tissue at 14 and 30 dpi. Our results demonstrate a recovery of the disease after an initial acute phase but also show persistence within the lumen and surrounding lymphoid tissue. These findings are relevant to developing effective control strategies.

Regulatory role of microRNA in mesenteric lymph nodes after Salmonella Typhimurium infection.

Salmonellosis is a gastrointestinal disease caused by non-typhoidal Salmonella serovars such as Salmonella Typhimurium. This pathology is a zoonosis, and food animals with subclinical infection constitute a vast reservoir for disease. After intestinal colonization, Salmonella Typhimurium reaches mesenteric lymph nodes (MLN), where infection is controlled avoiding systemic spread. Although the molecular basis of this infection has been extensively studied, little is known about how microRNA (miRNA) regulate the expression of proteins involved in the Salmonella-host interaction. Using small RNA-seq, we examined expression profiles of MLN 2 days after infection with Salmonella Typhimurium, and we found 110 dysregulated miRNA. Among them, we found upregulated miR-21, miR-155, miR-150, and miR-221, as well as downregulated miR-143 and miR-125, all of them previously linked to other bacterial infections. Integration with proteomic data revealed 30 miRNA potentially regulating the expression of 15 proteins involved in biological functions such as cell death and survival, inflammatory response and antigenic presentation. The inflammatory response was found increased via upregulation of miRNA such as miR-21 and miR-155. Downregulation of miR-125a/b, miR-148 and miR-1 were identified as potential regulators of MHC-class I components PSMB8, HSP90B1 and PDIA3, respectively. Furthermore, we confirmed that miR-125a is a direct target of immunoproteasome component PSMB8. Since we also found miR-130 downregulation, which is associated with upregulation of HSPA8, we suggest induction of both MHC-I and MHC-II antigen presentation pathways. In conclusion, our study identifies miRNA that could regulate critical networks for antigenic presentation, inflammatory response and cytoskeletal rearrangements.

Comparative proteomic analysis reveals different responses in porcine lymph nodes to virulent and attenuated homologous African swine fever virus strains.

African swine fever (ASF) is a pathology of pigs against which there is no treatment or vaccine. Understanding the equilibrium between innate and adaptive protective responses and immune pathology might contribute to the development of strategies against ASFV. Here we compare, using a proteomic approach, the course of the in vivo infection caused by two homologous strains: the virulent E75 and the attenuated E75CV1. Our results show a progressive loss of proteins by day 7 post-infection (pi) with E75, reflecting tissue destruction. Many signal pathways were affected by both infections but in different ways and extensions. Cytoskeletal remodelling and clathrin-endocytosis were affected by both isolates, while a greater number of proteins involved on inflammatory and immunological pathways were altered by E75CV1. 14-3-3 mediated signalling, related to immunity and apoptosis, was inhibited by both isolates. The implication of the Rho GTPases by E75CV1 throughout infection is also evident. Early events reflected the lack of E75 recognition by the immune system, an evasion strategy acquired by the virulent strains, and significant changes at 7 days post-infection (dpi), coinciding with the peak of infection and the time of death. The protein signature at day 31 pi with E75CV1 seems to reflect events observed at 1 dpi, including the upregulation of proteosomal subunits and molecules described as autoantigens (vimentin, HSPB1, enolase and lymphocyte cytosolic protein 1), which allow the speculation that auto-antibodies could contribute to chronic ASFV infections. Therefore, the use of proteomics could help understand ASFV pathogenesis and immune protection, opening new avenues for future research.

CE method for analyzing Salmonella typhimurium in water samples.

Salmonella typhimurium is commonly described as a food-borne pathogen. However, natural and drinking water are known to be important sources for the transmission of this pathogen in developing and developed countries. The standard method to determine Salmonella is laborious and many false positives are detected. To solve this, the present work was focused on the development of a capillary zone electrophoresis method coupled to ultraviolet detection for determination of Salmonella typhimurium in water (mineral and tap water). Separations were performed in less than 11 minutes using 4.5 mM Tris (hydroxymethyl)-aminomethane, 4.5 mM boric acid and 0.1 mM ethylene diamine tetraacetate (pH 8.4) with 0.1% v/v poly ethylene oxide as separation buffer. The precision of the method was evaluated in terms of repeatability obtaining a relative standard deviation of 10.5%. Using the proposed method Salmonella typhimurium could be separated from other bacteria that could be present in water such as Escherichia coli. Finally, the proposed methodology was applied to determine Salmonella typhimurium in tap and mineral water.

Transcriptional analysis of porcine intestinal mucosa infected with Salmonella Typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum.

Infected pork meat is an important source of non-typhoidal human salmonellosis. Understanding of molecular mechanisms involved in disease pathogenesis is important for the development of therapeutic and preventive strategies. Thus, hereby we study the transcriptional profiles along the porcine intestine during infection with Salmonella Typhimurium, as well as post-transcriptional gene modulation by microRNAs (miRNA). Sixteen piglets were orally challenged with S. Typhimurium. Samples from jejunum, ileum and colon, collected 1, 2 and 6 days post infection (dpi) were hybridized to mRNA and miRNA expression microarrays and analyzed. Jejunum showed a reduced transcriptional response indicating mild inflammation only at 2 dpi. In ileum inflammatory genes were overexpressed (e.g., IL-1B, IL-6, IL-8, IL1RAP, TNFα), indicating a strong immune response at all times of infection. Infection also down-regulated genes of the FXR pathway (e.g., NR1H4, FABP6, APOA1, SLC10A2), indicating disruption of the bile acid absorption in ileum. This result was confirmed by decreased high-density lipoprotein cholesterol in serum of infected pigs. Ileal inflammatory gene expression changes peaked at 2 dpi and tended to resolve at 6 dpi. Furthermore, miRNA analysis of ileum at 2 dpi revealed 62 miRNAs potentially regulating target genes involved in this inflammatory process (e.g., miR-374 and miR-451). In colon, genes involved in epithelial adherence, proliferation and cellular reorganization were down-regulated at 2 and 6 dpi. In summary, here we show the transcriptional changes occurring at the intestine at different time points of the infection, which are mainly related to inflammation and disruption of the bile acid metabolism.

Live attenuated African swine fever viruses as ideal tools to dissect the mechanisms involved in viral pathogenesis and immune protection.

African swine fever virus (ASFV) is the causal agent of African swine fever, a hemorrhagic and often lethal porcine disease causing enormous economical losses in affected countries. Endemic for decades in most of the sub-Saharan countries and Sardinia, the risk of ASFV-endemicity in Europe has increased since its last introduction into Europe in 2007. Live attenuated viruses have been demonstrated to induce very efficient protective immune responses, albeit most of the time protection was circumscribed to homologous ASFV challenges. However, their use in the field is still far from a reality, mainly due to safety concerns. In this study we compared the course of the in vivo infection caused by two homologous ASFV strains: the virulent E75 and the cell cultured adapted strain E75CV1, obtained from adapting E75 to grow in the CV1 cell-line. Interestingly, the kinetics of both viruses not only differed on the clinical signs that they caused and in the virus loads found, but also in the immunological pathways activated throughout the infections. Furthermore, E75CV1 confirmed its protective potential against the homologous E75 virus challenge and allowed the demonstration of poor cross-protection against BA71, thus defining it as heterologous. The in vitro specificity of the CD8(+) T-cells present at the time of lethal challenge showed a clear activation against the homologous virus (E75) but not against BA71. These findings will be of utility for a better understanding of ASFV pathogenesis and for the rational designing of safe and efficient vaccines against this virus.

Porcine sst1 can physically interact with other somatostatin receptors, and its expression is regulated by metabolic/inflammatory sensors.

The majority of the biological actions attributed to somatostatin (SST) are thought to be mediated by SST receptor 2 (sst2), the most ubiquitous sst, and, to a lesser extent, by sst5. However, a growing body of evidence suggests a relevant role of sst1 in mediating SST actions in (patho)physiological situations (i.e., endometriosis, type 2 diabetes). Moreover, sst1 together with sst2 and sst5 is involved in the well-known actions of SST on pituitary somatotropes in pig and primates. Here, we cloned the porcine sst1 (psst1) and performed a structural and functional characterization using both primary and heterologous models. The psst1 sequence presents the majority of signature motifs shared among G protein-coupled receptors and, specifically, among ssts and exhibits a high homology with other mammalian sst1, with only minor differences in the amino-terminal domain, reinforcing the idea of an early evolutive divergence between mammalian and nonmammalian sst1s. psst1 is functional in terms of decreasing cAMP levels in response to SST when transfected in heterologous models. The psst1 receptor is expressed in several tissues, and analyses of gene cis elements predict regulation by multiple transcription factors and metabolic stimuli. Finally, psst1 is coexpressed with other sst subtypes in various tissues, and in vitro data demonstrate that psst1 can interact with itself forming homodimers and with other ssts forming heterodimers. These data highlight the functional importance of sst1 on the SST-mediated effects and its functional interaction with different ssts, which point out the necessity of exploring the consequences of such interactions.

Fine-tuning of post-weaning pig microbiome structure and functionality by in-feed zinc oxide and antibiotics use.

Introduction: Post-weaning diarrhoea (PWD) is a multifactorial disease that affects piglets after weaning, contributing to productive and economic losses. Its control includes the use of in-feed prophylactic antibiotics and therapeutic zinc oxide (ZnO), treatments that, since 2022, are no longer permitted in the European Union due to spread of antimicrobial resistance genes and pollution of soil with heavy metals. A dysbiosis in the microbiota has been suggested as a potential risk factor of PWD onset. Understanding pig's microbiota development around weaning and its changes in response to ZnO and antibiotics is crucial to develop feasible alternatives to prophylactic and metaphylactic antimicrobial use. Methods: This study used shotgun metagenomic sequencing to investigate the environmental and faecal microbiota on 10 farms using (Treated) or not using (ZnO-free) in-feed antibiotics and ZnO during the first 14 days post-weaning (dpw). Environmental samples from clean pens were collected at weaning day (0dpw), and faecal samples at 0, 7 and 14dpw. Diarrhoeic faecal samples were collected at 7dpw when available. Results: The analysis of data revealed that the faecal microbiota composition and its functionality was impacted by the sampling time point (microbiota maturation after weaning) but not by the farm environment. Treatment with antibiotics and ZnO showed no effects on diversity indices while the analyses of microbiota taxonomic and functional profiles revealed increased abundance of taxa and metabolic functions associated with Phascolarctobacterium succinatutens or different species of Prevotella spp. on the Treated farms, and with Megasphaera elsdenii and Escherichia coli on the ZnO-free farms. The analysis of diarrhoea samples revealed that the treatment favoured the microbiota transition or maturation from 0dpw to 14dpw in Treated farms, resembling the composition of healthy animals, when compared to diarrhoea from ZnO-free farms, which were linked in composition to 0dpw samples. Discussion: The results provide a comprehensive overview of the beneficial effects of ZnO and antibiotics in PWD in the microbiota transition after weaning, preventing the overgrowth of pathogens such as pathogenic E. coli and revealing the key aspects in microbiota maturation that antibiotics or ZnO alternatives should fulfil.

Effects of removing in-feed antibiotics and zinc oxide on the taxonomy and functionality of the microbiota in post weaning pigs.

BACKGROUND: Post weaning diarrhoea (PWD) causes piglet morbidity and mortality at weaning and is a major driver for antimicrobial use worldwide. New regulations in the EU limit the use of in-feed antibiotics (Ab) and therapeutic zinc oxide (ZnO) to prevent PWD. New approaches to control PWD are needed, and understanding the role of the microbiota in this context is key. In this study, shotgun metagenome sequencing was used to describe the taxonomic and functional evolution of the faecal microbiota of the piglet during the first two weeks post weaning within three experimental groups, Ab, ZnO and no medication, on commercial farms using antimicrobials regularly in the post weaning period. RESULTS: Diversity was affected by day post weaning (dpw), treatment used and diarrhoea but not by the farm. Microbiota composition evolved towards the dominance of groups of species such as Prevotella spp. at day 14dpw. ZnO inhibited E. coli overgrowth, promoted higher abundance of the family Bacteroidaceae and decreased Megasphaera spp. Animals treated with Ab exhibited inconsistent taxonomic changes across time points, with an overall increase of Limosilactobacillus reuteri and Megasphaera elsdenii. Samples from non-medicated pigs showed virulence-related functions at 7dpw, and specific ETEC-related virulence factors were detected in all samples presenting diarrhoea. Differential microbiota functions of pigs treated with ZnO were related to sulphur and DNA metabolism, as well as mechanisms of antimicrobial and heavy metal resistance, whereas Ab treated animals exhibited functions related to antimicrobial resistance and virulence. CONCLUSION: Ab and particularly ZnO maintained a stable microbiota composition and functionality during the two weeks post weaning, by limiting E. coli overgrowth, and ultimately preventing microbiota dysbiosis. Future approaches to support piglet health should be able to reproduce this stable gut microbiota transition during the post weaning period, in order to maintain optimal gut physiological and productive conditions.

Pyroptosis and adaptive immunity mechanisms are promptly engendered in mesenteric lymph-nodes during pig infections with Salmonella enterica serovar Typhimurium.

In this study, we explored the transcriptional response and the morphological changes occurring in porcine mesenteric lymph-nodes (MLN) along a time course of 1, 2 and 6 days post infection (dpi) with Salmonella Typhimurium. Additionally, we analysed the expression of some Salmonella effectors in tissue to complete our view of the processes triggered in these organs upon infection. The results indicate that besides dampening apoptosis, swine take advantage of the flagellin and prgJ expression by Salmonella Typhimuriun to induce pyroptosis in MLN, preventing bacterial dissemination. Furthermore, cross-presentation of Salmonella antigens was inferred as a mechanism that results in a rapid clearance of pathogen by cytotoxic T cells. In summary, although the Salmonella Typhimurium strain employed in this study was able to express some of its major virulence effectors in porcine MLN, a combination of early innate and adaptive immunity mechanisms might overcome virulence strategies employed by the pathogen, enabling the host to protect itself against bacterial spread beyond gut-associated lymph-nodes. Interestingly, we deduced that clathrin-mediated endocytosis could contribute to mechanisms of pathogen virulence and/or host defence in MLN of Salmonella infected swine. Taken together, our results are useful for a better understanding of the critical protective mechanisms against Salmonella that occur in porcine MLN to prevent the spread of infection beyond the intestine.

Salmonella Typhimurium induces genome-wide expression and phosphorylation changes that modulate immune response, intracellular survival and vesicle transport in infected neutrophils.

Salmonella Typhimurium is a food-borne pathogen that causes salmonellosis. When in contact with the host, neutrophils are rapidly recruited to act as first line of defense. To better understand the pathogenesis of this infection, we used an in vitro model of neutrophil infection to perform dual RNA-sequencing (both host and pathogen). In addition, and given that many pathogens interfere with kinase-mediated phosphorylation in host signaling, we performed a phosphoproteomic analysis. The immune response was overall diminished in infected neutrophils, mainly JAK/STAT and toll-like receptor signaling pathways. We found decreased expression of proinflammatory cytokine receptor genes and predicted downregulation of the mitogen-activated protein (MAPK) signaling pathway. Also, Salmonella infection inhibited interferons I and II signaling pathways by upregulation of SOCS3 and subsequent downregulation of STAT1 and STAT2. Additionally, phosphorylation of PSMC2 and PSMC4, proteasome regulatory proteins, was decreased in infected neutrophils. Cell viability and survival was increased by p53 signaling, cell cycle arrest and NFkB-proteasome pathways activation. Combined analysis of RNA-seq and phosphoproteomics also revealed inhibited vesicle transport mechanisms mediated by dynein/dynactin and exocyst complexes, involved in ER-to-Golgi transport and centripetal movement of lysosomes and endosomes. Among the overexpressed virulence genes from Salmonella we found potential effectors responsible of these dysregulations, such as spiC, sopD2, sifA or pipB2, all of them involved in intracellular replication. Our results suggest that Salmonella induces (through overexpression of virulence factors) transcriptional and phosphorylation changes that increases neutrophil survival and shuts down immune response to minimize host response, and impairing intracellular vesicle transport likely to keep nutrients for replication and Salmonella-containing vacuole formation and maintenance.

SARS-CoV-2 accessory proteins involvement in inflammatory and profibrotic processes through IL11 signaling.

SARS-CoV-2, the cause of the COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome. Their roles during infection are still not completely understood. In this study, transcriptomics analysis revealed that both WNT5A and IL11 were significantly up-regulated in A549 cells expressing individual accessory proteins ORF6, ORF8, ORF9b or ORF9c from SARS-CoV-2 (Wuhan-Hu-1 isolate). IL11 is a member of the IL6 family of cytokines. IL11 signaling-related genes were also differentially expressed. Bioinformatics analysis disclosed that both WNT5A and IL11 were involved in pulmonary fibrosis idiopathic disease and functional assays confirmed their association with profibrotic cell responses. Subsequently, data comparison with lung cell lines infected with SARS-CoV-2 or lung biopsies from patients with COVID-19, evidenced altered profibrotic gene expression that matched those obtained in this study. Our results show ORF6, ORF8, ORF9b and ORF9c involvement in inflammatory and profibrotic responses. Thus, these accessory proteins could be targeted by new therapies against COVID-19 disease.

Using Shotgun Sequencing to Describe the Changes Induced by In-Feed Zinc Oxide and Apramycin in the Microbiomes of Pigs One Week Postweaning.

Postweaning diarrhea (PWD) is a relevant problem associated with early weaning on pig farms. For decades, in-feed antibiotics and therapeutic zinc oxide (ZnO) have been widely used to prevent PWD in piglets. The European Union is banning both strategies in 2022 due to antimicrobial resistance and environmental contamination concerns, respectively. Understanding the effects of these products on the pig microbiome is crucial for correcting potential microbial disbalances that would prompt PWD. Using shotgun sequencing, three trials were carried out to explore the impact of in-feed apramycin and ZnO, combined with different farm hygiene protocols, on the fecal microbiomes of piglets 7 days postweaning. In trial 1, 28-day-old piglets were allocated to one of three groups: control diet (Ct), Ct + ZnO (Zn), and Ct + apramycin (Ab). In trials 2 and 3, piglets were allocated to the same treatments, but the trials also included different cleaning protocols, achieving different hygiene levels. In-feed treatments impacted the richness, diversity, and relative abundance of the piglets' microbiome more than hygiene. Pigs in the Ct group showed higher species richness than pigs in the Ab and Zn groups. A clustering analysis evidenced a link between Enterobacteriaceae in the Ct group; Lactobacillaceae and Veillonellaceae mainly in the Ct group; and Bacteroidaceae, Ruminococcaceae, Oscillospiraceae, Acidaminococcaceae, and Lactobacillaceae in the Ab and Zn groups. Functional data analysis revealed a higher abundance of virulence genes in the Ct group microbiomes and heavy metal and antimicrobial resistance-related functions in the Zn treatment group. The results demonstrate that alternatives to Ab and ZnO should balance the microbial abundance and stimulate the growth of commensals to outcompete potential pathogens. IMPORTANCE Weaning is a critical period for piglets, during which potentially harmful bacteria such as Escherichia coli can increase in abundance in the intestine, creating digestive problems and diarrhea. In-feed antibiotics, the most frequent administration route for antibiotics in livestock, and therapeutic doses of zinc oxide (ZnO) help to control diarrhea but prompt secondary problems such as antimicrobial resistance and soil pollution from heavy metals. Understanding how these strategies impact the gut microbiota is crucial for establishing health biomarkers and designing successful replacement strategies. Using shotgun sequencing, this study compares the microbiota of pigs after early weaning when treated with in-feed antibiotics, ZnO, or treatment-free diets to describe differences that could define the susceptibility to infections, providing the basis for future research on improving intestinal resilience through microbiota-based strategies.

CD9 expression in porcine blood CD4+ T cells delineates two subsets with phenotypic characteristics of central and effector memory cells.

In this report, we describe the characterization of a new monoclonal antibody, named 4H5CR4, against porcine CD9. Its use in combination with antibodies to CD4, CD8α, and 2E3 allows to distinguish at least five main CD4+ T cell subsets. Analysis on these subsets of CD45RA, CD27, CD29, CD95, CCR7, and SLA-DR markers depicts a progressive model of CD4+ T cell development. CD4+ 2E3+ CD8α- CD9- cells are the least differentiated population of naïve cells, whereas the CD4+ 2E3- CD8α+CD9+ and CD4+ 2E3- CD8α+ CD9- cells display phenotypic features of central and effector memory T helper cells, respectively. The latter subsets were able to produce IFN-γ after polyclonal activation with PMA/Ionomycin; however, in vitro virus-specific IFN-γ production of PBMCs collected at 38-44 days after pseudorabies virus vaccination was dominated by cells with a CD9+ phenotype. Therefore, CD9 appears to be a useful marker to investigate CD4+ T cell heterogeneity in swine.

Impairment of antiviral immune response and disruption of cellular functions by SARS-CoV-2 ORF7a and ORF7b.

SARS-CoV-2, the causative agent of the present COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome, and some have been implicated in facilitating infection and pathogenesis through their interaction with cellular components. Among these proteins, accessory protein ORF7a and ORF7b functions are poorly understood. In this study, A549 cells were transduced to express ORF7a and ORF7b, respectively, to explore more in depth the role of each accessory protein in the pathological manifestation leading to COVID-19. Bioinformatic analysis and integration of transcriptome results identified defined canonical pathways and functional groupings revealing that after expression of ORF7a or ORF7b, the lung cells are potentially altered to create conditions more favorable for SARS-CoV-2, by inhibiting the IFN-I response, increasing proinflammatory cytokines release, and altering cell metabolic activity and adhesion. Based on these results, it is plausible to suggest that ORF7a or ORF7b could be used as biomarkers of progression in this pandemic.

Quantitative proteomics by 2-DE, 16O/18O labelling and linear ion trap mass spectrometry analysis of lymph nodes from piglets inoculated by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome. However, little is known regarding the mechanism(s) underlying the pathogenesis of PCV2-induced disease and the interaction of the virus with the host immune system. Here, we present a proteomics study on inguinal lymph nodes of piglets inoculated with PCV2, in order to better understand the pathogenesis of postweaning multisystemic wasting syndrome and the pathways might be affected after infection. We used two proteomics strategies, 2-DE and 1-DE followed by (16)O/(18)O peptide labelling and peptide identification and quantification by MS. More than 100 proteins were found to be differentially regulated and the results obtained by the two strategies were fairly concordant but also complementary, the (18)O labelling approach being a more robust alternative. Analysis of these proteins by systems biology tools revealed the implication of acute phase response and NrF2-mediated oxidative stress, suggesting a putative role for these pathways in the pig immune response. Besides, CD81 was found to be up-regulated, suggesting a possible role in the internalization of the virus. The use of proteomics technologies together with biology analysis systems opens up the way to gain more exhaustive and systematic knowledge of virus-pathogen interactions.

Immunohistochemical distribution of the tetraspanin CD9 in normal porcine tissues.

The tetra-membrane-spanning protein, CD9 is a 24-27 kDa cell surface glycoprotein expressed in a wide variety of human cells being involved in a variety of cell processes, including signaling, adhesion, motility, fertilization and tumor cells metastasis. By means of a polyclonal antibody (N1) raised against recombinant swine CD9 protein, we studied the immunohistochemical expression of CD9 on different normal swine tissues. Immunochemistry shows that swine CD9 was distribute in a similar form than in human tissues, being present on epithelial cells of lung, liver, kidney, skin, tonsil, testis (epididymo), gut mucosa, uterus and mama. Furthermore, polyclonal antibody against swine CD9 reacts with white matter from cerebrum and cerebellum, peripheral nerves fibers and Hassal corpuscle from thymus and ovum. Platelets react strongly with our antibody, but monocytes and neutrophils react lightly. These results suggest that CD9 antigen should play a similar functional role in swine and human and therefore studies on CD9 on swine as an animal model would allow new knowledge about its role in adhesion, fertilization and tumor metastasis among other important biomedical processes.

SARS-CoV-2 Accessory Proteins in Viral Pathogenesis: Knowns and Unknowns.

There are still many unanswered questions concerning viral SARS-CoV-2 pathogenesis in COVID-19. Accessory proteins in SARS-CoV-2 consist of eleven viral proteins whose roles during infection are still not completely understood. Here, a review on the current knowledge of SARS-CoV-2 accessory proteins is summarized updating new research that could be critical in understanding SARS-CoV-2 interaction with the host. Some accessory proteins such as ORF3b, ORF6, ORF7a and ORF8 have been shown to be important IFN-I antagonists inducing an impairment in the host immune response. In addition, ORF3a is involved in apoptosis whereas others like ORF9b and ORF9c interact with cellular organelles leading to suppression of the antiviral response in infected cells. However, possible roles of ORF7b and ORF10 are still awaiting to be described. Also, ORF3d has been reassigned. Relevant information on the knowns and the unknowns in these proteins is analyzed, which could be crucial for further understanding of SARS-CoV-2 pathogenesis and to design strategies counteracting their actions evading immune responses in COVID-19.